Adam P. Kohm

Cutting Edge: CD4+CD25+ Regulatory T Cells Suppress Antigen-Specific Autoreactive Immune Responses and Central Nervous System Inflammation During Active Experimental Autoimmune Encephalomyelitis

Autoreactive CD4+ T cells exist in normal individuals and retain the capacity to initiate autoimmune disease. The current study investigates the role of CD4+CD25+ T-regulatory (TR) cells during autoimmune disease using the CD4+ T cell-dependent myelin oligodendrocyte glycoprotein (MOG)-specific experimental autoimmune encephalomyelitis model of multiple sclerosis. In vitro, TR cells effectively inhibited both the proliferation of and cytokine production by MOG35–55-specific Th1 cells. In vivo, adoptive transfer of TR cells conferred significant protection from clinical experimental autoimmune encephalomyelitis which was associated with normal activation of autoreactive Th1 cells, but an increased frequency of MOG35–55-specific Th2 cells and decreased CNS infiltration. Lastly, transferred TR cells displayed an enhanced ability to traffic to the peripheral lymph nodes and expressed increased levels of the adhesion molecules ICAM-1 and P-selectin that may promote functional interactions with target T cells. Collectively, these findings suggest that TR cells contribute notably to the endogenous mechanisms that regulate actively induced autoimmune disease.

Cutting Edge: Anti-CD25 Monoclonal Antibody Injection Results in the Functional Inactivation, Not Depletion, of CD4+CD25+ T Regulatory Cells

CD4+CD25+ T regulatory (TR) cells are an important regulatory component of the adaptive immune system that limit autoreactive T cell responses in various models of autoimmunity. This knowledge was generated by previous studies from our lab and others using TR cell supplementation and depletion. Contrary to dogma, we report here that injection of anti-CD25 mAb results in the functional inactivation, not depletion, of TR cells, resulting in exacerbated autoimmune disease. Supporting this, mice receiving anti-CD25 mAb treatment display significantly lower numbers of CD4+CD25+ T cells but no change in the number of CD4+FoxP3+ TR cells. In addition, anti-CD25 mAb treatment fails to both reduce the number of Thy1.1+ congenic CD4+CD25+ TR cells or alter levels of CD25 mRNA expression in treatment recipients. Taken together, these findings have far-reaching implications for the interpretation of all previous studies forming conclusions about CD4+CD25+ TR cell depletion in vivo.

Cutting Edge: Ligation of the Glucocorticoid-Induced TNF Receptor Enhances Autoreactive CD4+ T Cell Activation and Experimental Autoimmune Encephalomyelitis

The glucocorticoid-induced TNFR (GITR) is expressed at high levels on resting CD4+CD25+ T regulatory (TR) cells and regulates their suppressive phenotype. Accordingly, we show that anti-GITR mAb treatment of SJL mice with proteolipid protein 139–151-induced experimental autoimmune encephalomyelitis significantly exacerbated clinical disease severity and CNS inflammation, and induced elevated levels of Ag-specific T cell proliferation and cytokine production. Interestingly, prior depletion of TR cells failed to result in exacerbated experimental autoimmune encephalomyelitis suggesting alternative targets for the anti-GITR mAb treatment. Importantly, naive CD4+CD25− T cells up-regulated GITR expression in an activation-dependent manner and anti-GITR mAb treatment enhanced the level of CD4+ T cell activation, proliferation, and cytokine production in the absence of TR cells both in vivo and in vitro. Taken together, these findings suggest a dual functional role for GITR as GITR cross-linking both inactivates TR cells and increases CD4+CD25− T cell effector function, thus enhancing T cell immunity.

Treatment with nonmitogenic anti-CD3 monoclonal antibody induces CD4+ T cell unresponsiveness and functional reversal of established experimental autoimmune encephalomyelitis.

In vivo administration of anti-CD3 Ab induces both immune tolerance and undesirable side-effects resulting from nonspecific proinflammatory cytokine production. In the current study, we investigated the therapeutic potential of two structurally altered forms of the anti-CD3 Ab in ameliorating established experimental autoimmune encephalomyelitis. Administration of either a chimeric (NM-IgG3) or digestion product (NM-F(ab′)2) form of the anti-CD3 Ab during established experimental autoimmune encephalomyelitis conferred significant protection from clinical disease progression and was associated with decreased Ag-specific T cell proliferation, cytokine production, and CNS inflammation. Interestingly, while this protection correlated with an increase in the frequency of CD4+CD25+ regulatory T cells, neither prior depletion of regulatory T cells nor anti-TGF-β treatment abrogated the treatment’s efficacy. Importantly, both treatments induced normal levels of intracellular Ca2+-flux, but significantly diminished levels of TCR signaling. Consequent to this decreased level of TCR-mediated signaling were alterations in the level of apoptosis and CD4+ T cell trafficking resulting in a profound lymphopenia. Collectively, these results indicate that nonmitogenic anti-CD3 directly induces a state of immune unresponsiveness in primed pathogenic autoreactive effector cells via mechanisms that may involve the induction of T cell tolerance, apoptosis, and/or alterations in cell trafficking.

Mimicking the way to autoimmunity: an evolving theory of sequence and structural homology

Although the etiology of autoimmune diseases remains largely unknown, a prevailing theory concerns the infection-induced activation of self-reactive lymphocytes via the process of molecular mimicry. Here, we discuss the theory of molecular mimicry and its continued evolution from the initial basic considerations of sequence similarity to the current theories of structural homology. Such findings serve to further our understanding of T-cell receptor degeneracy and might one day provide a direct link between infection and autoimmune disease.

Activation of Antigen-Specific CD4+ Th2 Cells and B Cells In Vivo Increases Norepinephrine Release in the Spleen and Bone Marrow

The neurotransmitter norepinephrine (NE) binds to the β2-adrenergic receptor (β2AR) expressed on various immune cells to influence cell homing, proliferation, and function. Previous reports showed that NE stimulation of the B cell β2AR is necessary for the maintenance of an optimal primary and secondary Th2 cell-dependent Ab response in vivo. In the present study we investigated the mechanism by which activation of Ag-specific CD4+ Th2 cells and B cells in vivo by a soluble protein Ag increases NE release in the spleen and bone marrow. Our model system used scid mice that were reconstituted with a clone of keyhole limpet hemocyanin-specific Th2 cells and trinitrophenyl-specific B cells. Following immunization, the rate of NE release in the spleen and bone marrow was determined using [3H]NE turnover analysis. Immunization of reconstituted scid mice with a cognate Ag increased the rate of NE release in the spleen and bone marrow 18–25 h, but not 1–8 h, following immunization. In contrast, immunization of mice with a noncognate Ag had no effect on the rate of NE release at any time. The cognate Ag-induced increase in NE release was partially blocked by ganglionic blockade with chlorisondamine, suggesting a role for both pre- and postganglionic signals in regulating NE release. Thus, activation of Ag-specific Th2 cells and B cells in vivo by a soluble protein Ag increases the rate of NE release and turnover in the spleen and bone marrow 18–25 h after immunization.

Sympathetic nervous system interaction with the immune system

Publisher Summary Both T cells and B cells express the β 2 -adrenergic receptor (AR) and bind norepinephrine that is released within lymphoid organs. Norepinephrine plays a role in modulating the activity of CD 4 + T cells and B cells participating in an immune response against antigen. This role of norepinephrine in regulating T and B cell activity needs to be fully understood because antibodies preserve well-being by defending against bacteria, viruses, and allergens and cytokines provide help to B cells, allowing the B cell to differentiate and thus secrete antibodies of particular isotypes. Given the importance to the host of having T cells and B cells that function optimally, it is likely that the mechanisms regulating and modulating these functions are varied and interrelated. For example, it is necessary to understand how products released from the hypothalamic-pituitary-adrenal axis, at the same time norepinephrine is released by sympathetic nerve terminals within lymphoid organs, affect immune cell function. An understanding of the mechanism by which the sympathetic nervous system (SNS) modulates the level of cytokines and antibody produced enables the development of therapeutic approaches for treating and preventing changes in the immunocompetent state of persons experiencing any disease that involves an alteration in either the nervous or the immune system function.

B Cell Receptor- and β2-Adrenergic Receptor-Induced Regulation of B7-2 (CD86) Expression in B Cells

The costimulatory molecule B7-2 (CD86) is expressed on the surface of APCs, including B cells. Considering the importance of B7-2 in regulating both T and B cell function, it may be important to understand the regulatory mechanisms governing its expression. We report in this study that stimulation of the B cell receptor (BCR) and/or a neurotransmitter receptor, the β2-adrenergic receptor (β2AR), may cooperate to regulate B cell-associated B7-2 expression in vitro and in vivo. β2AR stimulation further enhanced the level of BCR-induced B7-2 expression in B cells potentially via protein tyrosine kinase-, protein kinase A-, protein kinase C-, and mitogen-activated protein kinase-dependent mechanisms. Importantly, BCR and/or β2AR stimulation, but not histone hyperacetylation and DNA hypomethylation alone, increased B cell-associated B7-2 expression by increasing B7-2 mRNA stability, NF-κB nuclear binding, and NF-κB-dependent gene transcription. Thus, this study provides additional insight into the signaling intermediates and molecular mechanisms by which stimulation of the BCR and β2AR may regulate B cell-associated B7-2 expression.

Pathogenesis of NOD diabetes is initiated by reactivity to the insulin B chain 9-23 epitope and involves functional epitope spreading☆

Type 1 diabetes (T1D) is mediated by destruction of pancreatic β-cells by CD4 and CD8 T cells specific for epitopes on numerous diabetogenic autoantigens resulting in loss of glucose homeostasis. Employing antigen-specific tolerance induced by i.v. administration of syngeneic splenocytes ECDI cross-linked to various diabetogenic antigens/epitopes (Ag-SP), we show that epitope spreading plays a functional role in the pathogenesis of T1D in NOD mice. Specifically, Ag-SP coupled with intact insulin, Ins B(9-23) or Ins B(15-23), but not GAD65(509-528), GAD65(524-543) or IGRP(206-214), protected 4-6 week old NOD mice from the eventual development of clinical disease; infiltration of immune cells to the pancreatic islets; and blocked the induction of DTH responses in a Treg-dependent, antigen-specific manner. However, tolerance induction in 19-21 week old NOD mice was effectively accomplished only by Ins-SP, suggesting Ins B(9-23) is a dominant initiating epitope, but autoimmune responses to insulin epitope(s) distinct from Ins B(9-23) emerge during disease progression.

CCL22 regulates experimental autoimmune encephalomyelitis by controlling inflammatory macrophage accumulation and effector function

EAE is a demyelinating disease of the CNS and serves as a mouse model of MS. Expression of CCL22 in the draining LNs and spinal cord correlated with the onset of clinical EAE development and remained elevated. Administration of anti‐CCL22 at the time of autoantigen immunization delayed the initiation of clinical disease and dampened the severity of peak initial disease and relapses. Reduced EAE severity correlated with the reduction of pathology and leukocytes in the CNS, particularly, activated CD11b+Ly6Chi macrophages. There were no differences in effector T cell‐proliferative responses or effector T cell IFN‐γ or IL‐17 responses. However, treatment at the onset of disease did not reduce disease progression. Treatment of adoptive T cell transfer recipient mice with anti‐CCL22 resulted in decreased clinical disease development accompanied by a decrease in CNS accumulation of CD11b+Ly6Chi macrophages. Neutralization of CCL22 resulted in a macrophage population whose effector cytokine expression consisted of decreased TNF and increased IL‐10, a phenotype more consistent with M2 macrophages. This was corroborated by in vitro cultures of macrophages with CCL22. These results suggest that CCL22 functions to regulate development of EAE through macrophage chemoattraction and effector function.